Field of the Invention
The present invention relates generally to the fields of carbon nanotube chemistry, radioimmunotherapy and other targeted therapies. More specifically, the present invention relates to functionalized single wall nanotube therapeutic compositions, the construction thereof and uses therefor.
Description of the Related Art
Single wall carbon nanotubes (SWNT) offer both unusual opportunities as well as challenges. SWNTs are scalable, while retaining their key properties thus allowing design of a platform suitable for different applications in vivo. In addition, toxicity and clearance are size- and composition-dependent, allowing flexibility of design to reduce possible adverse effects. SWNTs have enormous aspect ratio, which allows huge amplification of effecter function and altered kinetics of conjugated agents. Additionally, all the carbons atoms, i.e., ˜8000 per 100 nm, are on the surface and therefore available for functionalization allowing attachments of multiple functionalities, in large numbers, simultaneously. This allows simultaneous applications as needed, e.g., for signaling molecules or for therapeutic agents. SWNT have a regular and identically repeating structure which should allow construction of regular and repeating functionalization, as well as patterns of functions. Biological processes interacting with the modified SWNT are likely to recognize the multivalency and repetition as they do for other biological recognition systems.
SWNT are inert, stable, flexible, and non-immunogenic. Most larger molecules introduced into living organisms can be recognized by the immune system, and thereafter quickly neutralized upon re-injection, recognized by metabolic systems, and therefore rapidly degraded, or are unstable and denatured within the reducing, warm and physiological environment of the body and tumors. SWNT have important electronic properties. The development of biologic sensors, telemetry or crude decision-making devices that could function ex vivo or in vivo may be possible with modified SWNT.
While there are many potential advantages to the use of SWNT as a base nanomaterial, there are also possible hurdles and disadvantages. The chemistry necessary to efficiently solubilize SWNT is beginning to be described but the effects of these identifications on the chemical and electronic properties of the SWNT are not known.
Although, parent single walled carbon nanotubes, unmodified and in some instances unpurified, have been reported to be toxic, there has been no thorough study concerning the in vivo biological properties of solubilized SWNTs. For example parent SWNTs have been reported to be cytotoxic to human keratinocytes and were also shown to inhibit growth of embryonic rat-brain neuron cells (1). It has been demonstrated separately that parent SWNTs induced the formation of mouse-lung granulomas (2). It has also been reported that SWNTs inhibit the proliferation of human HEK293 cells, induce cell apoptosis and decrease adhesive ability of cells (3). However, for therapeutic and diagnostic applications SWNTs soluble in aqueous media possessing free pendent functionalities for subsequent attachment of drugs or imaging agents are more appropriate. To this effect derivatized SWNTs with pendent peptides or CpG motifs have been shown to cross cell membranes (3). However, at SWNT concentrations greater than 10 μM cell death was dramatic and no clear mechanism was cited (4). On the other hand, the uptake of solubilized SWNTs (0.05 mg/mL) by endocytosis into a range of cell lines, e.g., H160, Jurkat, CHO and 3T3 fibroblasts, has been reported and it has been demonstrated that the nanotubes are localized in the endosomes and were non-toxic (5).
SWNT are unique among solid-state materials in that every atom is on the surface and hence surface chemistry could therefore be critical to the physical properties of SWNTs and their applications. SWNTs with aspect ratio approaching 104-105, form a unique class of one-dimensional quantum confined structures exhibiting either semiconducting (sem-) or metallic (met-) behavior are of special interest. Based on their sem- and/or met-character, a number of devices such as field-effect transistors, single electron transistors and computational logic gates have been demonstrated (6).
Moreover, SWNTs have the highest known specific conductivity per unit mass providing the ability to facilitate direct electron-transfer with biomolecules, acting as molecular-scale electrical conduits, and generating unique designing nano-scale biosensors. Similarities between the size scales of enzymes and chemically shortened SWNTs may promote the likelihood of SWNTs to come within electron tunneling distance of enzyme redox sites, improving sensitivity for enzyme labels that generate signals by direct electron exchange and communicate with external data capture devices. Bridging nanotubes with biological systems, however, is a relatively unexplored area, with the exception of a few reports on nanotube probe tips for biological imaging, nonspecific binding (NSB) of proteins, functionalization chemistry for bioimmobilization on nanotube sidewalls, internalization and transport through cell membrane of solubilized SWNTs in in vitro cell cultures and peptide SWNT constructs for vaccines.
SWNTs as platforms for investigating surface-protein and protein-protein binding and its subsequent use in cancer immunotherapy has remained completely unexplored. Hence methodologies aimed at the functionalization of nanotubes with specific antibodies and tagging SWNTs with alpha-emitting elements followed by the evaluation of both their biodistribution and specificity in vivo are needed and would have significant therapeutic implications. Early bio-functionalization approaches have relied heavily on modifications of the sidewalls of SWNTs via noncovalent chemistry so as to preserve the sp2 nanotube structure and thus their electronic characteristics (7). The 1,3-dipolar cycloaddition of azomethine ylides with SWNTs results in amine functionalized SWNTs and confers both aqueous solubility and chemical functionalization. These amine functionalized SWNTs are compatible with a myriad of established bioconjugation techniques (8).
There is a significant need in the art for therapeutic methods utilizing functionalized and soluble SWNTs comprising targeting and therapeutic moieties. Specifically, the prior art is deficient in methods of radioimmunotherapy or other immunotherapy or chemotherapy to treat or to prevent a cancer utilizing SWNTs which are functional to target and deliver a therapeutic molecule in vivo. The present invention fulfills this long-standing need and desire in the art.